Cite this article: Garrett KB, Schott R, Peshock
L, Yabsley MJ (2018). Prevalence and diversity
of piroplasms and ticks in young raccoons and
an association of Babesia sensu stricto
infections with splenomegaly. Parasitology
Open 4,e12,1–9. https://doi.org/10.1017/
Received: 31 December 2016
Revised: 26 February 2018
Accepted: 28 February 2018
Babesia; infants; neonates; raccoons;
splenomegaly; ticks; tick-borne; transmission
route; vertical transmission
Author for correspondence:
Kayla Garrett and Michael Yabsley, E-mail:
email@example.com and firstname.lastname@example.org
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Prevalence and diversity of piroplasms and
ticks in young raccoons and an association of
Babesia sensu stricto infections
Kayla Buck Garrett1,2, Renee Schott3, Lea Peshock4and Michael J. Yabsley1,2
Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA;
Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of
Georgia, Athens, GA 30602, USA;
Wildlife Rehabilitation Center of Minnesota, 2530 Dale St N, Roseville, MN 55113,
Greenwood Wildlife Rehabilitation Center, 5761 Ute Hwy, Longmont, CO 80503, USA
Piroplasms are intraerythrocytic parasites that are often transmitted by ixodid ticks, but vertical
transmission is an alternative route for some species. In the USA, raccoons (Procyon lotor)are
hosts for two known species, a Babesia microti-like sp. and Babesia lotori (in Babesia sensu
stricto group). To better understand the natural history of Babesia in raccoons, we tested
young raccoons from Minnesota and Colorado for Babesia spp., examined them for ticks,
and assessing for splenomegaly as a sign of clinical disease. Raccoons from both states were
infected with B. microti-like sp. and Babesia sensu stricto spp. Infections of B. microti-like
were common, even in 1-week-old raccoons, suggesting vertical transmission. Babesia sensu
stricto infections were more common in older raccoons. Raccoons infected with Babesia
sensu stricto had significantly higher spleen:body weight ratios compared with uninfected or
B. microti-like sp.-infected raccoons. Ticks were only found on raccoons from Minnesota.
The most common and abundant tick was Ixodes texanus but Ixodes scapularis and
Dermacentor variabilis were also found on raccoons. We report piroplasm infections and infes-
tations with several tick species in very young raccoons. Young raccoons infected with Babesia
sensu stricto spp. had higher spleen:body weight ratios, suggesting a disease risk.
The piroplasms are an important cause of disease in humans, domestic animals and some
wildlife, although most piroplasms in wildlife demonstrate low pathogenicity for their natural
host (Hunfeld et al. 2008; Yabsley and Shock, 2012). Most piroplasms with known life cycles
use ixodid ticks as vectors (Hunfeld et al. 2008) although vertical transmission has been
noted as a possible alternative transmission route for some piroplasms (e.g., Babesia microti
in laboratory mice and humans, Babesia gibsoni and Babesia canis canis in dogs and
Babesia bovis in cows) (Yeruham et al. 2003; Fukumoto et al. 2005; Joseph et al. 2012;
Mierzejewska et al. 2014; Bednarska et al. 2015; Adaszek et al. 2016; Costa et al. 2016). In add-
ition, fighting and intermixing of individuals’blood has been associated with direct transmis-
sion of B. gibsoni between fighting dogs (Yeagley et al. 2009).
Babesia infections in raccoons have been reported sporadically throughout the Eastern and
Midwestern USA (Schaffer et al. 1978; Anderson et al. 1981; Telford and Forrester, 1991;
Birkenheuer et al. 2006,2007; Clark et al. 2012). However, there are currently little data on
the pathogenicity of piroplasm infections in raccoons. A survey of raccoons from Japan
with splenomegaly, a pathologic consequence of Babesia infections in other species, found
that 8% (2/24) were positive for Babesia spp.; however, only raccoons with splenomegaly
were tested (Kawabuchi et al. 2005; Adaszek et al. 2016). Reports of Babesia infections in rac-
coons are generally based on older studies that utilized blood smears for piroplasms detection
(Schaffer et al. 1978; Anderson et al. 1981; Telford and Forrester, 1991).
Two species of morphologically similar, but molecularly distinct, piroplasms have been
reported from Florida, Massachusetts, North Carolina and Illinois (Goethert and Telford,
2003; Birkenheuer et al. 2006; Birkenheuer et al. 2007; Clark et al. 2012). One is a species
related to B. microti (hereafter called B. microti-like species) and the other is a Babesia
sensu stricto species now referred to as B. lotori (also called Babesia sp. AJB-2006)
(Anderson et al. 1981; Birkenheuer et al. 2007). The phylogenetic relationships of the piro-
plasms are under debate, but the B. microti clade is considered by many researchers to be a
novel genus and likely has many unique biological characteristics (Lack et al. 2012; Schreeg
et al. 2016). Without molecular characterization, it is unknown which or both of these species
are present in infected raccoons. Furthermore, the prevalence and distribution of these two
raccoon piroplasms is poorly known. Outside of the USA, via molecular assays, a low preva-
lence of B. microti-like parasites and at least one Babesia sensu stricto species have been
reported from raccoons introduced to Japan (Kawabuchi et al. 2005; Jinnai et al. 2009).
Currently, the transmission route is unknown for both Babesia
spp. of raccoons but it is presumed to be via ixodid ticks as most
piroplasms are transmitted by ixodid ticks (Uilenberg, 2006;
Hunfeld et al. 2008). Raccoons are commonly infested with sev-
eral tick species including Ixodes texanus,Ixodes cookei,Ixodes
scapularis,Ixodes affinis,Dermacentor variabilis,Amblyomma
americanum and A. maculatum, but the geographic distribution
and seasonality of many of these tick species varies (Dennis
et al. 1994; Ouellette et al. 1997; Yabsley et al. 2008). In North
Carolina, both piroplasms are found in raccoons in high preva-
lences (Birkenheuer et al. 2006), so the vector, if there is one, is
presumed to be a common tick species found on raccoons.
Unfortunately, all of the tick species noted above are found in
North Carolina so testing of raccoons from various parts of
North America would be needed to better identify possible tick
vectors (Ouellette et al. 1997; Birkenheuer et al. 2006).
However, there is the possibility of alternative transmission routes.
Numerous questions remain regarding the natural history of
piroplasm infections in raccoons, including the prevalence and
diversity of different piroplasm species infecting raccoons,
modes of transmission and pathogenicity. Thus, our objectives
were to determine the prevalence of Babesia in young raccoons
under the age of 6 weeks old and, if they occurred, what species
of Babesia were present. We hypothesized that young raccoons
would be infected with at least the two species of piroplasms pre-
viously reported in raccoons as the prevalence of both are very
high in adult raccoons. In addition, we calculated a spleen:body
weight ratio to determine if raccoons infected with Babesia spp.
had larger spleens because splenomegaly has been associated
with piroplasm infections. Finally, we examined the raccoons to
determine if an infestation of ticks occurred while still in the
nest, as raccoons younger than 6 weeks old typically do not ven-
ture from the nest (Schneider et al. 1971; Gehrt and Fritzell,
1998). The current vector(s) of any piroplasms of raccoons are
unknown but if piroplasm infections were noted in young rac-
coons, I. texanus, a tick species that is transmitted among rac-
coons in the nest (Anderson et al. 1981), would be expected to
be present and would possibly be associated with transmission.
However, it is also possible that other tick species may be trans-
mitted to young raccoons within the nest environment. We
sampled young raccoons in Minnesota and Colorado where we
have previously detected Babesia infections in adult raccoons
(Garrett and Yabsley, unpublished data).
From April to November of 2016, samples were collected from
fetal, neonatal or juvenile raccoons admitted to two rehabilitation
facilities: the Wildlife Rehabilitation Center of Minnesota
(Roseville, MN) and the Greenwood Wildlife Rehabilitation
Center in Colorado (Longmont, CO). The raccoons sampled
were presented to the centres deceased, died while in care, or
were euthanized due to poor prognosis. Raccoons were frozen
immediately after death to ensure no decomposition occurred
and shipped to the Southeastern Cooperative Wildlife Disease
Study (Athens, GA) where they were processed. Raccoons were
examined for ectoparasites, which if found, were preserved in
70% ethanol until identification. Ticks were identified morpho-
logically using published keys (Keirans and Litwak, 1989;Durden
and Keirans, 1996;Guglielmoneet al. 2014) or by using molecular
methods as described below. Spleens were removed after examin-
ation, weighed and re-frozen at −20 °C until testing.
Data collected from each raccoon included weight, body
length, sex and estimated age based on tooth eruption
(Montgomery, 1964). For a limited number of raccoons, age
was approximated based on weight because of missing or
damaged teeth. The age of remaining raccoons was also estimated
based on weight and both methods provided similar results (data
not shown). Although no animals were euthanized for the pur-
poses of this study, the collection of biological samples for patho-
gen testing was reviewed and approved by UGA’s Institutional
Animal Care and Use Committee (A2014 10–018).
Genomic DNA was extracted from ∼10 mg of spleen using a
commercial kit per the manufacturer’s instructions (DNEasy
Blood and Tissue kit, Qiagen, Hilden, Germany). Two different
PCR (polymerase chain reaction) assays targeting the V4 region
of the 18S rRNA gene of Babesia were used as described
(Birkenheuer et al. 2003,2007). One set of primers, BMlikeF
(5′-CTGCCTTATCATTAATTTCGCTTCCGAACG) and 793–
772R (5′-ATGCCCCCAACCGTTCCTATTA), targets Babesia
parasites in the B. microti-like clade. Molecular analyses were con-
ducted on a BioRad DNA Engine Peltier Thermal Cycler
(Bio-Rad Laboratories Incorporated, Foster City, CA). Cycling
parameters were 94 °C for 5 min followed by 49 cycles of 94 °C
for 45 s, 56 °C for 45 s and 72 °C for 45 s, with a final extension
of 72 °C for 5 min. The other set of primers, 455–479F
(5′-GTCTTGTAATTGG-AATGATGGTGAC) and 793–772R,
were used to detect Babesia sensu stricto species. Cycling para-
meters were 94 °C for 3 min followed by 44 cycles of 94 °C
for 30 s, 60 °C for 30 s, 72 °C for 30 s, with a final extension of
72 °C for 5 min.
Precautions were taken to prevent and detect contamination
including the performance of DNA extraction, PCR reaction
setup, and product analysis in distinct, designated areas.
Negative water controls were included in each set of DNA extrac-
tions. For each batch of PCR reactions, the extraction negative
control, a new water negative control and a positive control
(DNA sample from a pooled blood sample with the sequenced-
confirmed presence of B. lotori and B. microti-like sp.) were
included. Amplicons were observed in a GelRed stained 1.5%
agarose gel. Gels were run for an extended period of time to
ensure the reliable distinction between amplicon sizes.
Because the screening PCR assays amplify a small amplicon not
ideal for species identification, especially within the Babesia sensu
stricto group, 11 samples positive with the Babesia sensu stricto
group 18S screening primer set were also tested using a PCR target-
ing the cytochrome c oxidase subunit 1 (cox1) region and products
were sequenced to identify species present. Primers Babcox1F
(5′-GGAAGTGGWACWGG-WTGGAC) and Babcox1R (5′-TTC
GGTATTGCATGCCTTG) were used and cycling parameters
were 95 °C for 5 min followed by 45 cycles of 95 °C for 20 s,
50 °C for 30 s, 68 °C for 1 min and 30 s, and a final extension of
72 °C for 5 min (Schreeg et al. 2016). For eight samples that
were coinfected and a Babesia sensu stricto sequence was not
obtained using the 18S screening or cox1 gene protocols, we con-
ducted an additional PCR using Babesia sensu stricto-specific pri-
mers that target two regions of the large subunit rRNA (LSU)
gene fragment (lsu5 and lsu4)(Qurolloet al. 2017).
To confirm the presence of B. microti-like sp. in a subset of
samples the BMlikeF/793–772R amplicon was sequenced (nine
samples) or a larger region of the 18S rRNA gene was amplified
with primers 522F and 1661R and sequenced (four samples)
(Birkenheuer et al. 2007).
Amplicons were purified from an agarose gel using a gel-
purification kit (Qiagen) and bi-directionally sequenced at the
University of Georgia Genomics Facility (Athens, GA). Sequences
were cleaned using the Geneious program (Biomatters Limited,
2 Kayla Buck Garrett et al.
Auckland New, Zealand) and consensus sequences compared with
other Babesia sequences in GenBank.
Some Ixodes ticks were damaged during removal and could
not be identified to species using morphologic characteristics.
Tick DNA was extracted and amplified as described (Gleim
et al. 2014). Primers 16S −1(5
CAAGT) and 16S + 2 (5′-TTGGGCAAGAAGACCCTATGAA)
targeting the 16S rRNA gene were used and cycling parameters
were 94 °C for 2 min followed by 40 cycles of 94 °C for 30 s,
45 °C for 30 s, 72 °C for 1 min and a final extension of 72 °C
for 5 min. Amplicons were sequenced as described above.
A Fisher’s exact test was used to compare the prevalence of the
B. microti-like sp. and Babesia sensu stricto. In order to determine
the relationships between parasite prevalence and the different
variables, a generalized linear model (GLM) was utilized and
the data log transformed. The variables measured included sex,
age (by week), spleen:body weight ratio and whether or not
ticks were present. A separate GLM was performed for the
B. microti-like sp. and Babesia sensu stricto datasets.
A total of 106 young raccoons from Minnesota (n= 83) and
Colorado (n= 23) were included in the study and 66% (70/106)
were infected with Babesia. The prevalence of B. microti-like sp.
[66/106 (62%)] was significantly higher than that of Babesia
sensu stricto [11/106 (10%) (P= 0.0001)]. A total of eight
(7.5%) raccoons had coinfections. For the B. microti-like sp.,
infections were detected in individuals as young as 1 week of
age and prevalence was high in all age groups (Table 1). For the
Babesia sensu stricto group, infections were first noted at 2
weeks of age but prevalence was low, while prevalence in raccoons
that were 6 weeks or older was 40% (Table 1). Table 2 shows the
data for the 59 raccoons from Minnesota that were admitted in
identifiable litters. In general, the prevalence of Babesia within lit-
ters was high but for most litters, not all individuals were infected
Eight of the 11 Babesia sensu stricto samples amplified with the
cox1 PCR protocol but only five provided good quality sequences
(all from Minnesota). One sequence was most similar (99.8%) to a
Babesia sp. reported from a captive maned wolf (Chrysocyon bra-
chyurus) (KR017881) but was also 98.3% similar to Babesia lotori
(accessioned as Babesia sp. AJB-2006, KR017882) of raccoons.
Two other sequences, from the same litter of raccoons, were iden-
tical to each other and, although they were most similar to
B. lotori, they only shared 84.2% similarity (Table 2). The remain-
ing two sequences were most similar (90.4%) to B. vulpes (a
B. microti-like sp. of fox and dogs, accessed in GenBank as
Babesia sp. MES-2012, KC207827); however, these sequences
were identical to unpublished sequences of the B. microti-like
sp. of raccoons (Garrett and Yabsley, unpublished data). Both of
these raccoons were coinfected with a Babesia sensu stricto
based on the screening 18S PCR; one (16–5935) of which had a
partial 18S sequence to confirm Babesia sensu stricto infection
(Table 3). All eight of the raccoons that were coinfected samples
were successfully amplified using the LSU rRNA gene PCR assay.
These LSU sequences were (97.3–99.7%) similar to Babesia lintan
(Table 3). However, based on sequences from the cox1, 18S rRNA
and LSU gene sequences, there were three distinct groups of
Babesia sensu stricto (Table 3). Unique cox1 and 18S rRNA
gene sequences were submitted to GenBank (accession numbers
Many of the samples positive for the B. microti-like sp. using
the screen PCR did not produce amplicons with the cox1 PCR
protocol, so to confirm infections with this species, we sequenced
amplicons from the two different 18S PCR protocols. Near full-
length 18S rRNA sequences were acquired for four raccoons
and shorter 18S rRNA sequences were obtained for six additional
raccoons, including the three raccoons in the 1-week-old litter
(Table 2). All of these sequences were identical or most similar
(>99.5%) to the B. microti-like sp. sequences from raccoons avail-
able in GenBank (AB197940, AB935335 and AY144701).
Three tick species were found on young raccoons including
I. texanus,I. scapularis and D. variabilis, all from raccoons
from Minnesota (Table 4). Damaged ticks that could not be iden-
tified to species based on morphological characteristics were iden-
tified as I. texanus using PCR and sequence analysis. All life stages
of I. texanus were detected while only larvae and nymphs of I. sca-
pularis were found. Infestation with I. texanus was first noted on
raccoons at 2 weeks of age and the infestation prevalence was
similar for all age groups (Fig. 1A). In contrast, I. scapularis
Table 1. Prevalence of Babesia spp. in young raccoons from Colorado and Minnesota, by age class
Age in weeks no. positive/no. tested (% positive)
State Parasite <1
1 2 3 4 5 6+ Total
0/3 3/3 (100) 9/19 (47) 13/25 (52) 13/18 (72) 3/5 (60) 9/10 (90) 50/83 (60)
0/3 0/3 0/19 3/25 (12) 1/18 (6) 1/5 (20) 3/10 (30) 8/83 (10)
Coinfected 0/3 0/3 0/19 2/25 (8) 0/18 0/5 3/10 (30) 5/83 (6)
Colorado B. microti-like sp. NT 3/3 (100) 1/3 (33) 1/3 (33) NT 3/3 (100) 4/5 (80) 12/17 (70)
NT 0/3 1/3 (33) 0/3 NT 0/3 2/5 (40) 3/17 (18)
Coinfected NT 0/3 1/3 (33) 0/3 NT 0/3 2/5 (40) 3/17 (18)
Total B. microti-like sp. 0/3 6/6 (100) 10/22 (45) 14/28 (50) 13/18 (72) 6/8 (75) 13/15 (87) 62/100 (62)
0/3 0/3 1/22 (5) 3/28 (11) 1/18 (6) 1/8 (13) 5/15 (33) 11/100 (11)
Coinfected 0/3 0/3 1/22 (5) 2/28 (7) 0/18 0/8 5/15 (33) 8/100 (8)
Age was not available for six raccoons and were not included in the table.
These three raccoons were near-term and removed via caesarian section from a deceased female.
Parasitology Open 3
infestations were primarily noted in raccoons older than 5 weeks
of age, although a single 3-week-old raccoon was infested with
two I. scapularis nymphs (Fig. 1A). Infestation prevalence for
D. variabilis increased with age (Fig. 1A). The mean number of
I. texanus collected from infested raccoons was highest for rac-
coons in the 2-week age group with two individuals having 54
and 64 ticks, respectively (Fig. 1B). Other than those two raccoons
with high I. texanus infestations, tick burdens were generally low
with a maximum number of ticks collected from an individual
being three I. scapularis and 13 D. variabilis. Most ticks were
found in the ears or on the face (Fig. 2) although two raccoons
had ticks present on multiple parts of the body. Presence of
ticks was not a significant predictor variable for infection with
either piroplasm (B. microti-like sp.: P= 0.2393; Babesia sensu
Using GLM, the only significant variable for raccoons infected
with B. microti-like sp. was age (P= 0.0059). According to the
GLM, infection of raccoons with Babesia sensu stricto was asso-
ciated with age (P= 0.0017) and spleen:body weight ratio (P=
0.0005). Also, raccoons with Babesia sensu stricto infections had
significantly higher spleen:body weight ratios compared with rac-
coons infected with B. microti-like sp. only or those with no
Babesia infection (P= 0.0008 and P< 0.0001, respectively)
(Fig. 3). Although not significantly different from either group,
coinfected raccoons had increased average spleen:body ratio
(P= 0.374) (Fig. 3).
We detected Babesia infections in young raccoons from
Minnesota and Colorado with the B. microti-like sp. detected in
individuals as young as 1 week of age. There was a high prevalence
of the B. microti-like sp. in raccoons from Minnesota and
Colorado, and although Babesia sensu stricto infections were
detected, the prevalence was much lower. We obtained sequence
confirmation for the two piroplasms previously reported from
raccoons (a B. microti-like sp. and B. lotori), but we also found
possible novel Babesia spp. (groups B and C in Table 3). We
also noted coinfections occurring in some young raccoons; how-
ever, the prevalence of coinfection in both states was much lower
than previously reported for adult raccoons in North Carolina,
but this is likely due to the lower prevalence of Babesia sensu
stricto infections among the young raccoons we tested
(Birkenheuer et al. 2006). These data extend the known range
of B. lotori to Minnesota and confirms that Babesia sensu stricto
Table 2. Data on Babesia infections and ticks on raccoons from identifiable litters from Minnesota.
No. of infected
Species present based
on sequence analysis
No. of kits
No. of kits
infested with ticks B. microti-like sp.
1 Fetal 3 0 0 0 ND
2 1.5 5 5 (IT) 3 0 Three with B. microti-like
sp. (18S rRNA)
3 1.5–2.5 4 0 2 0 ND
4 1 3 0 3 0 Three B. microti-like sp.
52 5 0 2 0ND
6 1.5 2 2 (IT) 1 0 ND
7 2.5–3 5 1 (DV) 4 0 ND
8 4 3 2 (IT) 3 0 ND
9 2 3 1 (DV,IT) 1 0 B. microti-like sp. (18S
10 2.5 4 2 (IT) 1 2 Both confirmed Babesia
sensu stricto sp. (18S rRNA
One with B.
microti-like sp. (cox1)
11 4–5 2 0 2 2 Both with B. microti-like sp.
(18S rRNA); Both with
Babesia sensu stricto (LSU)
12 4–5 2 0 2 0 One with B. microti-like sp.
13 3 2 0 0 0 ND
14 3 3 3 (DV, IT) 1 0 ND
15 3 3 1 (IT) 1 0 ND
16 4–5.5 3 3 (IT) 2 0 ND
17 3–4 3 1 (IT) 3 0 ND
18 3–4 4 4 (DV, IT) 3 1 ND
IT, Ixodes texanus;DV,Dermacentor variabilis; Cox1, cytochrome c oxidase subunit I; LSU, large subunit rRNA; ND, not done.
Raccoons 16–4482 and 16–4484 in Table 3.
Raccoons 16–4596 and 16–4597 in Table 3.
4 Kayla Buck Garrett et al.
Table 3. Summary of PCR and sequence results for 11 raccoons positive for Babesia sensu stricto (eight coinfected with a B. microti-like sp. based on 18S rRNA screening PCR and three raccoons infected with only Babesia sensu
Cytochrome c oxidase
subunit 1 18S rRNA gene (long segment)
18S rRNA gene (short
segment, screening protocol) Large subunit rRNA gene
Neonates –coinfected 16–4596 A n.a.
99.4% (1013/1019) to Babesia sp. from
raccoon (AB197940) –aB. microti-like
99.1% (337/340) to Babesia sp.
from Japan (AB935172, AB251608)
98.1% (151/154) to B. lintan (KX698109)
16–4597 A n.a. 99.4% (478/481) to Babesia sp. from
raccoons (AB197940, AY144701,
AB935335) –aB. microti-like sp.
99.4% (326/328) to Babesia sp.
from Japan (AB935172, AB251608)
98.1% (151/154) to B. lintan (KX698109)
16–5713 B 99.8% (863/865) to a
Babesia sp. from a maned
n.a. 99.4% (153/154) to B. lintan (KX698109)
16–5935 B 90.4% [903/999] to B. vulpes
99.5% (551/554) to Babesia sp. from
raccoons (AB197940, AY144701,
AB935335) –aB. microti-like sp.
98.8% (327/331) to a Babesia sp.
from a maned wolf (KR017880)
99.7% (152/154) to B. lintan (KX698109)
16–5159 C 90.4% [856/949] to B. vulpes
98.7% (466/472) to Babesia sp. from
raccoons (AB197940, AY144701) –aB.
n.a. 97.3% (107/110) to B. lintan (KX698109)
CO-498 B n.a. n/d n.a. 99.4% (153/154) to B. lintan (KX698109)
CO-1350 B n.a. n/d n.a. 99.4% (153/154) to B. lintan (KX698109)
CO-861 B n.a. n/d n.a. 99.3% (151/152) to B. lintan (KX698109)
infected with Babesia
16–4484 A 84.2% (758/900) to B. lotori
n/d n.a. 98.1% (151/154) to B. lintan (KX698109)
16–5938 A n/a n/d 99.1% (338/341) to Babesia sp.
from Japan (AB935172, AB251608)
16–4482 A 84.2% (758/900) to B. lotori
n/d 99.4% (326/328) to Babesia sp.
from Japan (AB935172, AB251608)
Three groups of Babesia sensu stricto were identified based on sequences of the three gene targets.
n.a., not available (PCR either was negative or we were unable to get a clean sequence).
n/d, not done.
Parasitology Open 5
spp. occur in Minnesota and the B. microti-like sp. occurs in
Minnesota and Colorado.
The only previous study to investigate Babesia infections in
young racccons was conducted in Connecticut and our data sup-
port those findings (Anderson et al. 1981). Anderson et al. (1981)
found that three of four young raccoons were positive for Babesia
and nymphal I. texanus ticks were found on two of the raccoons.
However, in the previous study, infections in raccoons were deter-
mined based on blood smear analysis and thus the species of
Babesia present was unknown and the age of the raccoons was
not specified. Generally, we found a high prevalence of Babesia
among identifiable litters and although not all individuals in a lit-
ter were infected, these data were similar to data on vertical trans-
mission of B. microti in voles (Tolkacz et al. 2017).
A primary goal of our study was to determine if young rac-
coons were infected with Babesia and investigate the possible
role of vertical transmission as a route of infection. However,
because we also detected a high prevalence of tick infestation
on raccoons from Minnesota, it is unknown if the infections in
young raccoons were acquired vertically from infected female
Fig. 2. Ticks on a 1.5-week-old raccoon. (A) An adult Dermacentor variabilis on snout. (B) Several nymphal and adult Ixodes texanus in an ear. (C) Several adult I.
texanus in an ear.
Table 4. Number and stage of ticks collected from young raccoons from Minnesota
Tick species nLarvae Nymph Male Female
Ixodes texanus 211 7 133 1 70
Ixodes scapularis 93 6 0 0
Dermacentor variabilis 49 0 1 18 30
Fig. 1. (A) Per cent of young raccoon infested with ticks by age class in weeks (number of raccoons sampled in each age class show below age). (B) Average number
of ticks from infested raccoons in each age class in weeks. Number of raccoons sampled is the same as in (A).
Fig. 3. Effects of Babesia infection on average spleen:body weight ratio of young rac-
coons with standard error bars. Different letters denote significant differences among
6 Kayla Buck Garrett et al.
raccoons or were due to infestation with ticks at a very young age.
While we did not find ticks on raccoons younger than 2 weeks of
age, our sample size for 1-week-old raccoons was limited so it is
possible that raccoons become infested with ticks earlier than we
noted. We also did not note any infection in the three fetal rac-
coons; however, this was also a small sample size and samples
from the dam were not available for analysis.
The prepatent period is generally unknown for many piro-
plasms and varies by transmission route and detection method,
so reported data may not be valid for raccoon-infecting piro-
plasms. For vertical transmission of B. microti, voles in Europe
had a 3-week prepatent period and experimentally infected
BALB/c laboratory mice had a 20-day prepatent period
(Bednarska et al. 2015; Tolkacz et al. 2017). Our data suggest
that some Babesia infections may be acquired due to vertical
transmission, as we detected B. microti-like sp. infections in rac-
coons as young as 1 week old which is generally shorter than pre-
patent periods associated with tick transmission (e.g., 13–28 days
for B. microti to rhesus macaques (Macaca mulatta) and 13–17
days to BALB/c mice) (Ruebush et al. 1981;Liet al. 2016).
For Babesia sensu stricto spp., short prepatent periods after
exposure of hosts to infected ticks have been documented.
The prepatent period for B. canis transmitted to dogs by
Rhipicephalus sanguineus was 6 days post-infection, whereas cat-
tlebecameinfectedwithBabesia major within 9–15 days after
exposure to infected Haemaphysalis punctate (Paraense, 1949;
Yin et al. 1996). Vertical transmission of Babesia sensu stricto
sp. has also been noted with beagle puppies, whose mother
was intravenously inoculated with B. gibsoni prior to mating,
showing infection after 14 days (Fukumoto et al. 2005). Other
cases of vertical transmission of Babesia sensu stricto sp. have
been reported in puppies infected with Babesia canis 6 weeks
after birth and in puppies with clinical signs after 8 weeks
from presumed vertical transmission of B. canis canis
(Mierzejewska et al. 2014;Adaszeket al. 2016). Because infec-
tions of raccoons with Babesia sensu stricto were not noted
until at least 2–3 weeks of age, which is within the time frame
of tick-transmitted prepatent periods, it is possible that these
two groups of Babesia,B. microti-like sp. and Babesia sensu
stricto sp., utilize different transmission strategies.
Ticks, specifically ixodid ticks, are the presumed vectors for
Babesia sp. and are important to discuss when considering the
lifecycle of these raccoon piroplasms (Hunfeld et al. 2008). The
tick species found on our young raccoons from Minnesota are
commonly reported on raccoons (Dennis et al. 1994; Ouellette
et al. 1997; Hersh et al. 2012). Ixodes texanus was the most com-
mon and abundant tick found on the raccoons which was
expected as this species is found on host’s year-around and is
assumed to be acquired within the nests of their vertebrate
hosts (Sonenshine, 1993; Dharmarajan et al. 2016). This tick spe-
cies has a widespread distribution in the USA (Eastern and
Midwestern USA, California, and Alaska and likely many states
in between) (Ouellette et al. 1997; Gabriel et al. 2009; Durden
et al. 2016). The other two species found on our raccoons
included D. variablis (restricted to the Eastern USA and in iso-
lated populations in California) and I. scapularis (restricted to
the Eastern USA) (Bishopp and Trembley, 1945). Larval and
nymphal stages of I. scapularis feed on a wide range of small to
medium-sized hosts (mammals, birds, lizards), including rac-
coons, and this species is an important vector of Borrelia burgdor-
feri, the causative agent of Lyme disease, and B. microti, the
primary causative agent of human babesiosis in the USA
(Hersh et al. 2012). In our study, only larvae and nymphs of I.
scapularis were found on raccoons and in very low numbers,
most likely because of the earlier seasonal activity of these stages
compared with adults, which are more often found on hosts in fall
and winter (Bishopp and Trembley, 1945). Raccoons younger
than 6–8 weeks most likely acquire ticks from the mother, as
young of this age do not typically venture from the nest, while
older individuals (7–10-week-old young) are more active and
can become infested with ticks outside of the nest (Schneider
et al. 1971; Gehrt and Fritzell, 1998).
Piroplasm infections in most species of wildlife are considered
to be of low pathogenicity, although under certain circumstances
(e.g., coinfections, immunosuppression, stress, climate factors,
etc.) they may cause disease (Penzhorn, 2006; Yabsley and Shock,
2012). Examples include babesiosis in African lions suffering
from a concurrent canine distemper virus outbreak and decreased
food availability, and the development of fatal babesiosis in black
rhinoceros due to the stress of capture for translocation efforts
(Penzhorn, 2006;Munsonet al. 2008). In raccoons, Babesia infec-
tions are presumed to be of little clinical significance but most
reports are surveys of healthy free-ranging adults. One possible
clinical case in a raccoon was a single juvenile raccoon from
Illinois that was infected with B. lotori (Birkenheuer et al. 2006).
The raccoon was found non-ambulatory with pronounced
anaemia, hypoproteinaemia, hypalbuminaemia and elevated ala-
nine aminotransferase with rare intraerythrocytic Babesia parasites.
It was treated for Babesia and released; however, it is unknown if it
was the Babesia infection that caused the clinical signs or if they
were the results of a secondary infection (Birkenheuer et al.
2006). In general, clinical disease is likely to be more pronounced
in young animals. Studies on vertical transmission of piroplasms in
several hosts indicate that clinical signs in infected young generally
occur between 17 and 25 days (Fukumoto et al. 2005;Bednarska
et al. 2015;Brownet al. 2015;Adaszeket al. 2016). Because our
sampled animals were not available for antemortem testing, we
used the ratio of spleen weight:body weight as a measure of pos-
sible disease. The association with splenomegaly and Babesia
sensu stricto infections, but not with B. microti-like sp. infections,
suggests that early infections with B. lotori or the possible novel
Babesia sp. may cause clinical disease in young raccoons.
Unfortunately, because these raccoons were dead on arrival or
euthanized on entry, no clinical pathology data were collected
nor was any histologic analysis done to determine the cause of
death or illness (although many were admitted because they were
orphaned, not because they were sick). Splenomegaly is one of
many common findings of clinical babesiosis in many host species
(Kawabuchi et al. 2005; Mierzejewska et al. 2014;Solano-Gallego
et al. 2016), including puppies that acquired B. canis infection
through vertical transmission (Mierzejewska et al. 2014). Babesia
has been detected in raccoons with splenomegaly in Japan; how-
ever, only raccoons with splenomegaly were tested and the preva-
lence of Babesia was low, possibly because raccoons were
introduced to Japan (Kawabuchi et al. 2005;Jinnaiet al. 2009).
It is possible that Babesia spp. of certain wildlife may be more
pathogenic than currently recognized but only impact very young
animals that are rarely studied.
Because we obtained B. microti sequences with the cox1 PCR,
this protocol can amplify both Babesia sensu stricto species and B.
microti-like sp. Unfortunately, due to financial constraints, clon-
ing of these coinfected samples was not possible for this study.
However, there were no polymorphic bases present in the cox1
sequences although the sequences that failed may have been
due to mixed amplicons. We did not PCR test ticks collected
from raccoons for piroplasms because ticks were all potentially
blood-fed so any positives could have occurred from ingestion
of infected blood from the raccoon, the previous infection from
another raccoon, or vertical transmission of piroplasms within
infected ticks. One of our objectives was to investigate the possi-
bility of vertical transmission of piroplasms to raccoons but since
we detected ticks at a young age and dams were not available for
Parasitology Open 7
testing, we could not determine if transmission of piroplasms
occurred vertically or via tick transmission.
In summary, we show that several species of Babesia infect
very young raccoons. In addition, we showed that several species
of ticks are parasitizing these young raccoons, so it is currently
unknown if these infections are a result of vertical transmission
or tick-transmission due to early infestation. Finally, we note
that young raccoons infected with Babesia sensu stricto spp.
have a higher spleen:body weight ratio suggesting possible clinical
disease associated with infection. Additional studies are needed to
better understand the natural history, diversity and impact of
Babesia infections in raccoons.
Acknowledgements. We thank Chris Cleveland, Brianna Williams and
Maddy Pfaff for assistance with laboratory procedures and statistical analyses.
We also thank the staff at both wildlife rehabilitation centres for their assist-
ance with the project.
Financial assistance was provided by the Warnell School of Forestry
and Natural Resources (support of K.B.G.) and the sponsorship of
the Southeastern Cooperative Wildlife Disease Study by the fish
and wildlife agencies of Alabama, Arkansas, Florida, Georgia,
Kentucky, Kansas, Louisiana, Maryland, Mississippi, Missouri,
Nebraska, North Carolina, Ohio, Oklahoma, Pennsylvania, South
Carolina, Tennessee, Virginia and West Virginia, USA. Support
from the states to SCWDS was provided in part by the Federal
Aid to Wildlife Restoration Act (50 Stat. 917).
Conflict of interest
The authors assert that all procedures contributing to this work
comply with the ethical standards of the relevant national and
institutional guides on the care and use of laboratory animals.
Although no animals were euthanized for the purposes of this
study, the collection of biological samples for pathogen testing
was reviewed and approved by UGA’s Institutional Animal Care
and Use Committee (A2014 10-018).
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